Necrotizing enterocolitis (NEC) is a devastating disorder that affects approximately 10% of premature infants. Its mortality remains high (15-30%), and its cause remains unknown. About 80% of cases occur within 35 days of birth among hospitalized newborns of low birth weight. Probiotics diminish the incidence and severity of NEC, and NEC does not occur antepartum. NEC affects a readily identifiable at-risk group, has a tightly defined interval before its onset, occurs in an organ system that is intimately associated with a microbial population in flux, has a plausible association with the intestinal microbiota, and cohorts at risk have rarely been studied in large numbers, or prospectively. This disorder, therefore, provides a unique opportunity to explore the role of the human enteric microbiome in a devastating disease. Moreover, NEC epidemiology and age-incidence present an ability to enroll and study cohorts that are highly likely to provide valuable pathophysiologic and microbiologic insights. In this project, we will identify and quantify the microbial components of stool and its products before and at the onset of NEC. In doing so, we will test the overarching hypothesis that NEC is a direct or indirect consequence of the enteric biomass, its products, or both. We will use multicenter cohorts of premature infants at high risk of developing NEC, extend our research on this disease currently sponsored by the Washington University Institute of Clinical and Translational Sciences, and continue our longstanding collaborations with the Genome Center at Washington University and the Washington University Digestive Diseases Research Core Center (Informatics Core). The Aims of this proposal are to (1) conduct a case cohort study in which we compare clinical data and biological specimens from cases and well-matched controls; (2) determine if the kind and density of intestinal biomass, its gene content, and transcriptional activity are associated with, and potential determinants of, NEC; and (3) determine if host risk alleles for intestinal inflammation play a role in the development of NEC. These efforts will be accomplished using subjects from three collaborating neonatal intensive care units (NICUs), focusing on the critical, instructive, and understudied pre-NEC stage of illness, and formulating a data repository that will be a resource for investigators worldwide who wish to focus their efforts on NEC, its precipitants, and its prevention and cure.